Susceptibility to rifampicin
Of the 149 isolates tested by BACTEC 114 were found susceptible and 35 resistant to rifampicin at 2 μg/ml. The number of isolates found susceptible by the phage assay varied with the concentration of drug used. (Table 1). When tested at a drug concentration of 2 μg/ml 91 isolates were found susceptible and 58 resistant. Twenty three isolates found resistant by the phage assay at this drug concentration were found susceptible by BACTEC. However, when concentration of rifampicin was increased to 4 or 10 μg/ml the number of strains found resistant by the phage assay dropped to 39 and, when compared to BACTEC, four isolates with discordant results were recorded. The 35 isolates found resistant by BACTEC were also found resistant by the phage assay at all drug concentrations tested. Comparison of the phage assay when used at 4 or 10 μg/ml with the BACTEC provided an overall agreement for the two tests of 97.3% (n = 145/149, 95% CI; 93.3–99.3%). If the results from BACTEC are taken as the "gold standard" for assessing resistance and susceptibility, the sensitivity of the phage test was 100% (i.e. 35/35: lower 97.5% one sided CI = 90%) and the specificity of the phage test was 96.5% (i.e. 110/114: 95% CI 91.3–99.1%).
The statistical significance of the difference between the two assays can be assessed by comparing the number of discrepant results. For 4 isolates the phage assay indicated resistance and the BACTEC indicated susceptibility and there were no isolates which were susceptible by the phage test and resistant by BACTEC. This difference (4 vs 0) is not statistically significant. However, further genetic analysis (see below) suggested that the phage assay might be the more sensitive of the two. Phage assay results were available within 48 hours.
Mutation analysis
Sequencing of the rpoB core region was performed on the 39 isolates found resistant by the phage assay at 4 or 10 μg/ml and on 18 of the 19 isolates found resistant to 2 μg/ml but susceptible at 4 or 10 μg/ml.
All but one of the 39 isolates found resistant at 4 or 10 μg/ml in the phage assay had a rpoB mutation associated with RMP resistance (Table 2). A double mutation was found in two isolates. The isolate with no mutation in the rpoB core region was found resistant by both BACTEC and phage. All 18 isolates tested that were resistant to 2 μg/ml but susceptible to 4 μg/ml by phage displayed a wild type sequence for the rpoB core region. These 18 isolates were all susceptible by the BACTEC, suggesting their true susceptibility to rifampicin. The four isolates with discordant BACTEC and phage results at 4 or 10 μg/ml each harbored mutations predictive of resistance to rifampicin, two (both from the same patient) had Leu511Pro mutations and the other two had a Asp516Tyr and a Leu533Pro mutation.
Minimal inhibitory concentration (MIC)
MIC's of the four isolates found resistant by phage at 4 or 10 μg/ml of drug but susceptible by BACTEC were determined by the colorimetric MTT method. The MIC of each isolate was found to be greater than 50 μg/ml compared to 2 μg/ml for the reference susceptible isolate (H37Rv) and all four isolates were judged to be truly resistant, in agreement with the phage and sequence data.
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